Virtually every cell in the body is exposed to mechanical forces that are transduced through its cytoskeleton and influence its activity. Recent studies using T cells, the master organizing cells of the adaptive immune system, have shown that mechanical forces acting upon the T cell receptor (TCR) and through the cytoskeleton can trigger cellular responses. It is unclear how the cytoskeleton plays this role, or how T cells interpret the interplay of forces and signals of receptor ligation. We have discovered that T cells are more sensitive to antigen (i.e., have a lower threshold of activation) when their actin cytoskeleton is untethered and mechanically soft. Furthermore, we have discovered that in autoimmune diabetes, mechanical forces from inflamed extracellular matrix (ECM) can provoke T cell autoimmunity. The gaps in our knowledge are due to a lack of tools that can measure and deliver nanoscale forces to live cells. That T cells encode their threshold of activation in cytoskeletal structures provides impetus to study how cells communicate in a language that combines mechanical forces with receptor signals. Here, we propose to 1) Determine the mechanosensitivity of the T cell receptor at the molecular level, using an advanced atomic force microscope (AFM) to convey mechanical forces and antigens to single-molecule TCRs; 2) Identify the cytoskeletal networks required for mechanosensing, using a new generation of AFM cantilevers that can measure cytoskeletal changes in live cells; and 3) Determine influence of mechanical forces due to inflammatory ECM, using a mouse model of autoimmune diabetes and a 3D biomimetic matrix to emulate the inflamed ECM and its mechanical effects on T cell activation. Our lab, working with our collaborators at Stanford, has pioneered breakthroughs in biological atomic force microscopy (AFM) and nanofabrication that make these innovative studies possible by allowing us to precisely ligate receptors and exert minute forces while measuring mechanical responses in live T cells, all while imaging cells and their cytoskeletal changes using live confocal microscopy. These studies will help us decipher the language of mechanical forces in cell signaling, and will have a major impact on our understanding of the mechanical effects of inflammatory ECM in autoimmune diseases. We expect our findings will spur novel immune therapeutics. Our new methods of using AFM in cell biology have the potential to revolutionize studies of mechanobiology and receptor signaling pathways implicated in many diseases.